DNA replication is central to genome integrity and intimately tied to large-scale 3D chromosome organization and cell lineage specification, but a lack of tools with which to probe causality have limited progress in under- standing its regulation. In particular, there is a critical need to identify cis-elements regulating replication timing (RT) as a first step toward addressing causal linkages to chromosome architecture and gene regulation. Our long-term goal is to understand the relationship of RT to chromosome architecture, epigenetic states and disease. Our immediate goal is to identify elements regulating developmentally programmed changes in RT and examine their role in genome organization and transcription. Our central hypothesis is that discrete functional elements dictate developmentally programmed changes in RT independently from transcription. The rationale for this proposal is that identifying cis-elements regulating RT is essential to identify causal pathways linking RT to higher order chromosome folding and gene expression. Preliminary data establish feasibility to engineer genome deletions, inversions and ectopic insertions and identify DNA segments that are necessary and/or sufficient for RT regulation during murine embryonic stem cell (mESC) differentiation. We also provide evidence that transcription is neither necessary nor sufficient for RT changes. Aim1 will use CRIPR/Cas9-mediated chromosome engineering to generate further deletions and inversions within and between adjacent domains and evaluate their consequences to RT, TAD structure and sub-nuclear compartment. Aim2 will introduce cloned genomic and/or synthetically modified DNA sequences from a developmentally regulated replication domain into a constitutively replicated domain to delineate sequences sufficient to transfer developmental RT control to the ectopic site and to determine what aspects of 3D chromosome structure co-transfer with RT regulation. Aim3 will use these same tools to evaluate the extent to which cis-elements controlling transcription can regulate RT switches and vice versa. This contribution will be significant because identifying necessary and sufficient RT regulatory DNA sequences has not previously been possible and it is the essential first step toward a molecular understanding of RT developmental control and its links to chromosome architecture and, ultimately, human disease. The proposed work is innovative in combining novel facile chromosome engineering methods with well-characterized directed mESC differentiation systems to uncover pathways eliciting developmentally programmed changes in RT. This knowledge will have major impact on our understanding of RT and its relationship to 3D chromosome organization, it will guide future studies probing the significance of RT aberrations in human disease, and it will contribute innovations in chromosome domain engineering that will impact many facets of chromosome biology.